Patients with NF2 exhibit loss of function mutations or deletions the NF2 tumor suppressor gene. The NF2 gene encodes a 595 amino acid tumor suppressor protein called Merlin. Merlin has homology to the Ezrin, Radixin and Moesin (ERM) group of cellular proteins that link integral membrane proteins to the actin cytoskeleton. Rac and Cdc42, small GTPases of the Rho family of proteins, are critical regulators of the cytoskeleton and Merlin has been implicated as an inhibitor of Rac-dependent signaling. Merlin also inhibits cell growth. The binding partners for Merlin may be critically important in its growth suppressive function. We have identified a novel interaction between Merlin and Mixed Lineage Kinase 3 (MLK3). MLK3 is a serine/threonine kinase that activates multiple MAP kinase signaling pathways. Cdc42 and Rac are upstream activators of MLK3. Our studies suggest that Merlin is a physiological inhibitor of MLK3. We postulate that Merlin blocks Rac- dependent signaling by inhibiting MLK3 activation of MAPK signaling. Furthermore, the lack of functional Merlin in NF2 tumor cells could cause an increase in the basal level of active MLK3 which may promote cell growth and cellular transformation. The aims of this proposal are to analyze the functional significance of the interaction between Merlin and MLK3, to investigate if loss of NF2 expression augments MLK3, ERK and JNK kinase activities, and to determine if Merlin inhibits cell proliferation and characteristics of malignant transformation of MLK3-transformed cells and human tumor cells. We will perform co- immunoprecipitations of full length and truncated, overexpressed MLK3 and Merlin proteins to identify the specific regions of Merlin and MLK3 that are required for their interaction. Indirect immunofluorescence studies will be employed to study the colocalization of MLK3 and Merlin in schwann cells. Binding of MLK3 to its upstream activators will be analyzed in the presence or absence of co-expressed Merlin. Normal cells, tumor cells lacking NF2 expression, cells that have regulated expression of NF2, and cells in which NF2 expression has been silenced, will be used to analyze the effect of Merlin on MLK3, ERK and JNK activation. The impact of Merlin on MLK3- mediated cellular transformation will be investigated in MLK3-transformed NIH-3T3 cells, SKOV3 ovarian tumor cells and HEI-193 tumor cells. Cells will be infected with a lentivirus that overexpresses Merlin, or Merlin lacking the MLK3 binding region, and cell proliferation, anchorage independent growth, and invasiveness of the cells will be analyzed. Collectively, the results of this study will allow us to define the biochemical significance of the Merlin-MLK3 interaction and the impact of Merlin on MLK3-dependent signaling and cellular transformation. We have identified a novel interaction between Merlin, the protein product of the NF2 tumor suppressor gene, and MLK3, a MAP3K that regulates multiple MAPK pathways. This proposal is focused on deciphering the function of Merlin in regulation of MLK3- dependent MAPK signaling, cell proliferation and cellular transformation. Since silencing mlk3 inhibits proliferation of NF2 human schwannoma and NF2 -/- mouse osteosarcoma cells, MLK3 could be a promising new target for the development of therapies to treat NF2 tumors. Collectively, the results of this study will be an important contribution to our current understanding of Merlin function, will give us valuable insight into MAPK signaling in NF2, and will expand our knowledge of potential signaling proteins that can be targeted for the development of treatments for patients with NF2. [unreadable] [unreadable] [unreadable]